One approach for a detailed understanding of dynamical cellular processes during drug delivery is the use of functionalized biocompatible nanoparticles and fluorescent markers. An appropriate imaging system has to detect these moving particles so as whole cell volumes in real time with high lateral resolution in a range of a few 100 nm. In a previous study Extended depth-of-field microscopy (EDF-microscopy) has been applied to fluorescent beads and tradiscantia stamen hair cells and the concept of real-time imaging has been proved in different microscopic modes. In principle a phase retardation system like a programmable space light modulator or a static waveplate is incorporated in the light path and modulates the wavefront of light. Hence the focal ellipsoid is smeared out and images seem to be blurred in a first step. An image restoration by deconvolution using the known point-spread-function (PSF) of the optical system is necessary to achieve sharp microscopic images of an extended depth-of-field. This work is focused on the investigation and optimization of deconvolution algorithms to solve this restoration problem satisfactorily. This inverse problem is challenging due to presence of Poisson distributed noise and Gaussian noise, and since the PSF used for deconvolution exactly fits in just one plane within the object. We use non-linear Total Variation based image restoration techniques, where different types of noise can be treated properly. Various algorithms are evaluated for artificially generated 3D images as well as for fluorescence measurements of BPAE cells.
Extended depth-of-field (EDF) microscopy is a well-investigated and very simple method to obtain projection images with an extended depth of focus. Despite its advantages of being a real-time method applicable to any microscopic mode with high lateral resolution that can be simply realized by extending a commercial microscope, the lack of z-correlation is still a problem. In this work we present a combined technique of EDF and stereomicroscopy. By cross-correlation depth information is obtained. Finally, 3D images are reconstructed for best phase masks and simulation results are evaluated experimentally.
Pupil phase masks for enhanced depth of field microscopy were investigated by using a spatial light modulator. The
phase masks were evaluated with simulations in terms of the mean square error between in-focus and out-of-focus point
spread functions. The resulting best-performing phase masks were tested for fluorosphere samples using a microscope
add-on containing the SLM. First, z-stacks of fixed fluorospheres in an agarose medium were recorded in order to
measure the extended depth of field. The same measurements were also performed on fluorospheres subjected to
Brownian motion in an aqueous solution. The results show that with deconvolution and appropriate filtering it is possible
to obtain sharp fluorosphere images with an extended depth of field of at least 10 μm.
Real-time visualization of live-cell dynamic processes has been realized in differential interference contrast (DIC)
microscopy, with an extended-depth-of-focus (EDF) increase of about one order of magnitude. In addition, the
diffraction-limited lateral resolution of the microscope is preserved. Experimentally, a custom-designed waveplate
inserted in the optical path of a microscope causes feature information, from within the entire 3D specimen volume, to be
uniformly encoded into a single CCD image in a way that, after processing, defocus blur artifacts are removed. The
result is that extended-depth feature information can be visualized at video rates during live-cell dynamics investigations
because there is no longer the need to acquire multi-focus image stacks at each time point. Retrieving the encoded
extended-depth information requires specialized digital image processing techniques. This work concentrates on digital
filter design for the reconstruction of the waveplate-encoded images. As a measure of filter quality, the signal-to-noise
ratio (SNR), the modulation transfer function and the least mean square values are evaluated. Obtaining a high SNR and
a lateral resolution comparable to those in conventional single-focus-plane microscopy images at the same time is a
challenging goal in EDF microscopy. Filters are created in the frequency domain on the basis of the measured
waveplate-encoded point spread functions. Results show that it is possible to produce video-rate, extended-depth-offocus
images that have low noise levels and diffraction-limited resolution. This is illustrated by movies of fluorescent
beads and of cytoplasmic streaming in live stamen hair cells from the spiderwort plant, Tradescantia, using extendeddepth